AU2010288239B2 - Adjuvant comprising aluminium, oligonucleotide and polycation - Google Patents

Abstract

An immunological adjuvant comprises an aluminium salt, an immunostimulatory oligonucleotide and a polycationic polymer, wherein the oligonucleotide and the polymer ideally associate with each other to form a complex. The adjuvant can be included in a composition with an immunogen e.g. to elicit an immune response that protects against a bacterial disease or a fungal disease.

Description

WO 2011/024071 PCT/IB2010/002259 ADJUVANT COMPRISING ALUMINIUM, OLIGONUCLEOTIDE AND POLYCATION This application claims the benefit of US provisional application 61/237,595 filed August 27th 2009, the complete contents of which are hereby incorporated herein by reference for all purposes. TECHNICAL FIELD 5 This invention is in the field of vaccine adjuvants and their combinations. BACKGROUND ART Adjuvants are included in many current vaccines. Aluminium salts, typically aluminium hydroxide or aluminium phosphate, are by far the most common adjuvants. Although they are usually used as single adjuvants, they have also been combined with other non-aluminium adjuvants e.g. the 10 FENDRIXTM product includes an adjuvant of aluminium phosphate plus 3d-MPL, and the CERVARIXTM product's adjuvant is aluminium hydroxide plus 3d-MPL. It is an object of the invention to provide modified and improved combination adjuvants which include aluminium salts. DISCLOSURE OF THE INVENTION 15 The invention provides an immunological adjuvant comprising an aluminium salt, an immunostimulatory oligonucleotide and a polycationic polymer. The oligonucleotide and the polymer ideally associate with each other to form a complex. The invention also provides an immunogenic composition comprising (i) an adjuvant of the invention and (ii) an immunogen. The immunogen can be: adsorbed to aluminium salt in the 20 adjuvant; adsorbed to oligonucleotide/polymer complex in the adjuvant; and/or adsorbed to neither the aluminium salt nor complex. The invention also provides a process for preparing an immunological adjuvant of the invention, comprising a step of mixing an aluminium salt with a complex of an immunostimulatory oligonucleotide and a polycationic polymer. In alternative methods, the aluminium salt, 25 immunostimulatory oligonucleotide and polycationic polymer are mixed before the complex has formed. For example, the aluminium salt can be mixed with the oligonucleotide, and then the polymer is added; or the aluminium salt can be mixed with the polymer, and then the oligonucleotide is added. The complex may form after the oligonucleotide and the polymer meet. The invention also provides a process for preparing an immunogenic composition comprising a step 30 of mixing (i) an adjuvant of the invention and (ii) an immunogen. The immunogen, aluminium salt, oligonucleotide and polymer may be mixed in any order. For example, the invention provides a process for preparing an immunogenic composition of the invention, comprising a step of mixing (i) an aluminium salt and (ii) an immunogen; and then mixing the salt/immunogen mixture with an immunostimulatory oligonucleotide and a polycationic polymer. 35 The invention also provides a process for preparing an immunogenic composition of the invention,

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WO 2011/024071 PCT/IB2010/002259 comprising a step of mixing (i) an immunostimulatory oligonucleotide and a polycationic polymer, typically in the form of a complex, and (ii) an immunogen; and then mixing the oligonucleotide/polymer/immunogen mixture with an aluminium salt. In one preferred embodiment, an immunogen is adsorbed to an aluminium salt (e.g. an aluminium hydroxide adjuvant) and the 5 adsorbed immunogen is then mixed with oligonucleotide/cationic polymer complexes. The invention also provides a kit comprising: (i) a first container that contains an adjuvant of the invention; and (ii) a second container that contains an immunogen and/or a further adjuvant. The invention also provides a kit comprising: (i) a first container that contains an aluminium salt; and (ii) a second container that contains an immunostimulatory oligonucleotide and a polycationic polymer. 10 One or both of the first and second containers may include an immunogen. Thus the contents of the two containers can be combined (e.g. at the point of use) to form an adjuvant or immunogenic composition of the invention. These kits may include a third container that contains an immunogen and/or a further adjuvant. The aluminium salt 15 Adjuvants of the invention include at least one aluminium salt. Suitable aluminium salts include the adjuvants known individually as aluminium hydroxide and aluminium phosphate. These names are conventional, but are used for convenience only, as neither is a precise description of the actual chemical compound which is present [e.g. see chapter 9 of reference 1]. The invention can use any of the "hydroxide" or "phosphate" adjuvants that are in general use as adjuvants. The use of an 20 aluminium hydroxide adjuvant is preferred. The adjuvants known as "aluminium hydroxide" are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AlO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH) 3 . by infrared (IR) spectroscopy, in particular by the presence of an adsorption 25 band at 1070cm 1 and a strong shoulder at 3090-3100cm- 1 [chapter 9 of ref. 1]. The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as 30 seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. Mean particle diameters in the range of 1-1 Ojim are reported in reference 2. The pI of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al+ at pH 7.4 have been reported for aluminium hydroxide adjuvants. 35 The adjuvants known as "aluminium phosphate" are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation -2- WO 2011/024071 PCT/IB2010/002259 influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PO 4 /Al molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AlPO 4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164cm 1 (e.g. when heated to 200"C) indicates the presence of structural hydroxyls [chapter 9 of 5 ref. 1]. The PO 4 /Al" 3 molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95+0.1. The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /Al molar ratio between 0.84 and 0.92, included at 0.6mg Al 3 /ml. The aluminium phosphate will generally be particulate (e.g. plate-like morphology 10 as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5 20gm (e.g. about 5-1Opm) after any antigen adsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mg Al*** at pH 7.4 have been reported for aluminium phosphate adjuvants. The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions 15 and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate = more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7. 20 A mixture of both an aluminium hydroxide and an aluminium phosphate has can also be used. In this situation there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2:1 e.g. J5:1, >6:1, >7:1, 8:1, > 9: 1, etc. Thus an adjuvant of the invention may comprise: (i) an aluminium hydroxide, an immunostimulatory oligonucleotide and a polycationic polymer; (ii) an aluminium phosphate, an immunostimulatory 25 oligonucleotide and a polycationic polymer; or (iii) an aluminium hydroxide, an aluminium phosphate, an immunostimulatory oligonucleotide and a polycationic polymer. The concentration of AlI*I in a pharmaceutical composition of the invention will usually be <lOmg/ml e.g. <5 mg/ml, :4 mg/ml, <3 mg/ml, <2 mg/ml, <I mg/ml, etc. A preferred range is between 0.3 and 1 mg/ml. 30 The immunostimulatory oligonucleotide and the polycationic polymer The invention uses an immunostimulatory oligonucleotide and a polycationic polymer. These are ideally associated with each other to form a particulate complex, which usefully is a TLR9 agonist. Immunostimulatory oligonucleotides are known as useful adjuvants. They often contain a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked to a guanosine) and their 35 adjuvant effect is discussed in refs. 3-8. Oligonucleotides containing TpG motifs, palindromic sequences, multiple consecutive thymidine nucleotides (e.g. TTTT), multiple consecutive cytosine -3- WO 2011/024071 PCT/IB2010/002259 nucleotides (e.g. CCCC) or poly(dG) sequences are also known immunostimulants, as are double-stranded RNAs. Although any of these various immunostimulatory oligonucleotides can be used with the invention, it is preferred to use an oligodeoxynucleotide containing deoxyinosine and/or deoxyuridine [9], and ideally an oligodeoxynucleotide containing deoxyinosine and 5 deoxycytosine. Inosine-containing oligodeoxynucleotides may include a CpI motif (a dinucleotide sequence containing a cytosine linked to an inosine). The oligodeoxynucleotide may include more than one (e.g. 2, 3, 4, 5, 6 or more) CpI motif, and these may be directly repeated (e.g. comprising the sequence (CI),, where x is 2, 3, 4, 5, 6 or more) or separated from each other (e.g. comprising the sequence (CIN),, where x is 2, 3, 4, 5, 6 or more, and where each N independently represents one or 10 more nucleotides). Cytosine residues are ideally unmethylated. The oligonucleotides will typically have between 10 and 100 nucleotides e.g. 15-50 nucleotides, 20-30 nucleotides, or 25-28 nucleotides. It will typically be single-stranded. The oligonucleotide can include exclusively natural nucleotides, exclusively non-natural nucleotides, or a mix of both. For instance, it may include one or more phosphorothioate linkage(s), and/or one or 15 more nucleotides may have a 2'-O-methyl modification. A preferred oligonucleotide for use with the invention is a single-stranded deoxynucleotide comprising the 26-mer sequence 5'-(IC) 13 -3' (SEQ ID NO: 1). This oligodeoxynucleotide forms stable complexes with polycationic polymers to give a good adjuvant. The polycationic polymer is ideally a polycationic peptide, such as a cationic antimicrobial peptide. 20 The polymer may include one or more leucine amino acid residue(s) and/or one or more lysine amino acid residue(s). The polymer may include one or more arginine amino acid residue(s). It may include at least one direct repeat of one of these amino acids e.g. one or more Leu-Leu dipeptide sequence(s), one or more Lys-Lys dipeptide sequence(s), or one or more Arg-Arg dipeptide sequence(s). It may include at least one (and preferably multiple e.g. 2 or 3) Lys-Leu dipeptide sequence(s) and/or at 25 least one (and preferably multiple e.g. 2 or 3) Lys-Leu-Lys tripeptide sequence(s). The peptide may comprise a sequence R-XZXZxXZX-R 2 , wherein: x is 3, 4, 5, 6 or 7; each X is independently a positively-charged natural and/or non-natural amino acid residue; each Z is independently an amino acid residue L, V, I, F or W; and R, and R 2 are independently selected from the group consisting of -H, -NH 2 , -COCH 3 , or -COH. In some embodiments X-R 2 may be an amide, 30 ester or thioester of the peptide's C-terminal amino acid residue. See also reference 10. A polycationic peptide will typically have between 5 and 50 amino acids e.g. 6-20 amino acids, 7-15 amino acids, or 9-12 amino acids. A peptide can include exclusively natural amino acids, exclusively non-natural amino acids, or a mix of both. It may include L-amino acids and/or D-amino acids. L-amino acids are typical. -4- WO 2011/024071 PCT/IB2010/002259 A peptide can have a natural N-terminus (NH 2 -) or a modified N-terminus e.g. a hydroxyl, acetyl, etc. A peptide can have a natural C-terminus (-COOH) or a modified C-terminus e.g. a hydroxyl, an acetyl, etc. Such modifications can improve the peptide's stability. A preferred peptide for use with the invention is the 11-mer KLKLLLLLKLK (SEQ ID NO: 2; ref 5 11), with all L-amino acids. The N-terminus may be deaminated and the C-terminus may be hydroxylated. A preferred peptide is H-KLKL 5 KLK-OH, with all L-amino acids. This oligopeptide is a known antimicrobial [12], neutrophil activator [13] and adjuvant [14] and forms stable complexes with immunostimulatory oligonucleotides to give a good adjuvant. The most preferred mixture of immunostimulatory oligonucleotide and polycationic polymer is the 10 TLR9 agonist known as IC31TM [15-17], which is an adsorptive complex of oligodeoxynucleotide SEQ ID NO: 1 and polycationic oligopeptide SEQ ID NO: 2. The oligonucleotide and oligopeptide can be mixed together at various ratios, but they will generally be mixed with the peptide at a molar excess. The molar excess may be at least 5:1 e.g. 10:1, 15:1, 20:1, 25:1, 30;1, 35:1, 40:1 etc. A molar ratio of about 25:1 is ideal [18,19]. Mixing at this excess 15 ratio can result in formation of insoluble particulate complexes between oligonucleotide and oligopeptide. The complexes can be combined with an aluminium salt as described herein. The oligonucleotide and oligopeptide will typically be mixed under aqueous conditions e.g. a solution of the oligonucleotide can be mixed with a solution of the oligopeptide with a desired ratio. The two solutions may be prepared by dissolving dried (e.g. lyophilised) materials in water or buffer 20 to form stock solutions that can then be mixed. The complexes can be analysed using the methods disclosed in reference 20. Complexes with an average diameter in the range I gm-20gnm are typical. Poly-arginine and CpG oligodeoxynucleotides similarly form complexes [21]. The complexes can be maintained in aqueous suspension e.g. in water or in buffer. Typical buffers 25 for use with the complexes are phosphate buffers (e.g. phosphate-buffered saline), Tris buffers, Tris/sorbitol buffers, borate buffers, succinate buffers, citrate buffers, histidine buffers, etc. As an alternative, complexes may sometimes be lyophilised. Complexes in aqueous suspension can be centrifuged to separate them from bulk medium (e.g. by aspiration, decanting, etc.). These complexes can then be re-suspended in an alternative medium if 30 desired. Mixing of aluminium salt, oligonucleotide and polymer Adjuvant compositions of the invention can conveniently be prepared by mixing an aqueous suspension of the aluminium salt with an aqueous suspension of the oligonucleotide/polymer complex. The salt and complex are each typically maintained in liquid form, hence providing an easy 35 way of co-formulating them. -5- WO 2011/024071 PCT/IB2010/002259 In some embodiments one or both of the suspensions includes an immunogen so that the mixing provides an immunogenic composition of the invention. In other embodiments neither liquid includes an immunogen, so the mixed product (i.e. the adjuvant composition of the invention) can later be combined with an immunogen to provide an immunogenic composition of the invention. 5 Where two liquids are mixed the volume ratio for mixing can vary (e.g. between 20:1 and 1:20, between 10:1 and 1:10, between 5:1 and 1:5, between 2:1 and 1:2, etc.) but is ideally about 1:1. The concentration of components in the two suspensions can be selected so that a desired final concentration is achieved after mixing e.g. both may be prepared at 2x strength such that 1:1 mixing provides the final desired concentrations. 10 It is also possible to prepare the adjuvant composition in other ways e.g. by centrifuging the aluminium salt and then resuspending the pellet in a suspension of the complex, by centrifuging the complexes and then resuspending the pellet in a suspension of the aluminium salt, etc. Various concentrations of oligonucleotide and polycationic polymer can be used e.g. any of the concentrations used in references 15, 18, 19 or 22. For example, a polycationic oligopeptide can be 15 present at 1100 pM, 1000 pM, 350 pM, 220pM, 200 pM, 110 pM, 100 pM, 11 IM, 1OpM, 1pM, 500nM, 50nM, etc. An oligonucleotide can be present at 44 nM, 40 nM, 20nM, 14 nM, 4.4 nM, 4 nM, 2 nM, etc. A polycationic oligopeptide concentration of less than 2000 nM is typical. For SEQ ID NOs: 1 & 2, mixed at a molar ratio of 1:25, the concentrations in mg/mL in three embodiments of the invention may thus be 0.311 & 1.322, or 0.109 & 0.463, or 0.031 and 0.132. 20 An aluminium salt and a complex of the immunostimulatory oligonucleotide and polycationic polymer are typically both particulate. The mean particle diameter of aluminium salt adjuvants is typically in the order of 1-20pm [2,23]. This is also the size range for complexes seen in IC31TM. When such particles are combined, the average diameter of the salt particles may be substantially the same as the average diameter of the complexes. In other embodiments, however, the average 25 diameter of the salt particles may be smaller than the average size of the complexes. In other embodiments, the average diameter of the salt particles may be larger than the average size of the complexes. Where the average diameters differ, the larger diameter may be greater by a factor of at least 1.05x e.g. 1.1x, 1.2x, 1.3x, l.4x, 1.5x, 2x, 2.5x, 3x or more. If either the salt or the complex has particles with a range of diameters, but the average diameters differ, the ranges may or may not 30 overlap. Thus the largest salt particle may be smaller than the smallest complex particles, or the largest complex particles may be smaller than the smallest salt particles. Because the particles are generally too large to be filter sterilised, sterility of an adjuvant composition of the invention will typically be achieved by preparing the aluminium salt and the complex under sterile conditions, and then mixing them under sterile conditions. For instance, the components of the 35 complex could be filter sterilised, and then mixed under sterile conditions to form sterile complexes. These sterile complexes could then be mixed with an autoclaved (sterile) aluminium salt adjuvant to -6- WO 2011/024071 PCT/IB2010/002259 provide a sterile adjuvant composition. This sterile adjuvant can then be mixed with a sterile immunogen to give an immunogenic composition suitable for patient administration. The density of aluminium salt particles is typically different from the density of a complex of immunostimulatory oligonucleotide and polycationic polymer, which means that the two particles 5 might be separated based on density e.g. by sucrose gradient. A composition comprising a mixture of an aluminium salt, an immunostimulatory oligonucleotide and a polycationic polymer can also usefully include (i) a buffer, such as a histidine buffer e.g. 10mM histidine buffer and/or (ii) 5-15mg/ml sodium chloride e.g. 9mg/ml sodium chloride. A composition including a histidine buffer can usefully have a pH in the range of 6.0 and 7.4 10 e.g. between 6.3 and 7.0, or about 6.5. Pharmaceutical compositions Adjuvant compositions of the invention usually include components in addition to the aluminium salt, oligonucleotide and polymer e.g. they typically include one or more pharmaceutically acceptable component. Such components may also be present in immunogenic compositions of the 15 invention, originating either in the adjuvant composition or in another composition. A thorough discussion of such components is available in reference 24. A composition may include a preservative such as thiomersal or 2-phenoxyethanol. It is preferred that the vaccine should be substantially free from (e.g. <10pg/ml) mercurial material e.g. thiomersal free. Vaccines containing no mercury are more preferred. Preservative-free vaccines are particularly 20 preferred. a-tocopherol succinate can be included as an alternative to mercurial compounds in influenza vaccines. To control tonicity, a composition may include a physiological salt, such as a sodium salt. Sodium chloride (NaCI) is preferred, which may be present at between 1 and 20 mglml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or 25 magnesium chloride, etc. Compositions may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, maybe within the range of 280-330 mOsm/mg or 290-310 mOsm/kg. The pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8. 30 A composition is preferably sterile. A composition is preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. A composition is preferably gluten free. A useful composition comprises a histidine buffer (e.g. 10mM histidine buffer), sodium chloride (e.g. 9mg/ml sodium chloride) and an aluminium hydroxide adjuvant (e.g. 2mg/mi Al ). -7- WO 2011/024071 PCT/IB2010/002259 An immunogenic composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a 'multidose' kit). The inclusion of a preservative is useful in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for 5 removal of material. Compositions will generally be in aqueous form at the point of administration. Vaccines are typically administered in a dosage volume of about 0.5ml, although a half dose (i.e. about 0.25ml) may sometimes be administered e.g. to children. In some embodiments of the invention a composition may be administered in a higher dose e.g. about Iml e.g. after mixing two 0.5ml volumes. 10 Immunogens Adjuvant compositions of the invention can be administered to animals in combination with immunogens to induce an immune response. The invention can be used with a wide range of immunogens, for treating or protecting against a wide range of diseases. The immunogen may elicit an immune response that protects against a viral disease (e.g. due to an enveloped or non-enveloped 15 virus), a bacterial disease (e.g. due to a Gram negative or a Gram positive bacterium), a fungal disease, a parasitic disease, an auto-immune disease, or any other disease. The immunogen may also be useful in immunotherapy e.g. for treating a tumour/cancer, Alzheimer's disease, or an addiction. The immunogen may take various forms e.g. a whole organism, an outer-membrane vesicle, a protein, a saccharide, a liposaccharide, a conjugate (e.g. of a carrier and a hapten, or of a carrier and a 20 saccharide or liposaccharide), etc. The immunogen may elicit an immune response against an influenza virus, including influenza A and B viruses. Various forms of influenza virus immunogen are currently available, typically based either on live virus or on inactivated virus. Inactivated vaccines may be based on whole virions, split virions, or on purified surface antigens. Influenza antigens can also be presented in the form of 25 virosomes. Hemagglutinin is the main immunogen in current inactivated vaccines, and vaccine doses are standardised by reference to HA levels, typically measured by SRID. Existing vaccines typically contain about 15ig of HA per strain, although lower doses can be used e.g. for children, or in pandemic situations, or when using an adjuvant. Fractional doses such as /2 (i.e. 7.5 ig HA per strain), 4 and 1/8 have been used, as have higher doses (e.g. 3x or 9x doses [25,26]). Thus 30 compositions may include between 0.1 and 150pg of HA per influenza strain, preferably between 0.1 and 50pg e.g. 0.1-20pg, 0.1-15tg, 0.1-10ptg, 0.1-7.5pg, 0.5-5pig, etc. Particular doses include e.g. about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc. per strain. It is usual to include substantially the same mass of HA for each strain included in the vaccine e.g. such that the HA mass for each strain is within 10% of the mean HA mass per strain, 35 and preferably within 5% of the mean. For live vaccines, dosing is measured by median tissue culture infectious dose (TCID 50 ) rather than HA content, and a TCID 50 of between 106 and 108 (preferably between 1065-107-5) per strain is typical. Rather than use SPF eggs as the substrate for viral growth, -8- WO 2011/024071 PCT/IB2010/002259 where virus is harvested from infected allantoic fluids of hens' eggs, cell lines that support influenza virus replication may be used. The cell line will typically be of mammalian origin e.g. MDCK. Influenza A virus immunogens may be from any suitable HA subtype strain e.g. HI, H3, H5, H7, H9 etc., such as a HINI, H3N2 and/or H5N1 strain. 5 The immunogen may elicit an immune response against a Candida fungus such as C.albicans. For instance, the immunogen may be a p-glucan, which may be conjugated to a carrier protein. The glucan may include P-1,3 and/or p-1,6 linkages. Suitable immunogens include those disclosed in references 27 & 28. The immunogen may elicit an immune response against a Streptococcus bacterium, including 10 S.agalactiae, S.pneumoniae and S.pyogenes. For instance, the immunogen may be a capsular saccharide, which may be conjugated to a carrier protein. For S.agalactiae the saccharide may be from one or more of serotypes la, Ib, II, III, and/or V. For S.pneumoniae the saccharide may be from one or more of serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and/or 23F. In addition to (or in place of) capsular saccharide inmunogen(s), polypeptide immunogens may be used to elicit a protective 15 anti-streptococcal immune response e.g. comprising RrgB, as disclosed in reference 29. The immunogen may elicit an immune response against a Staphylococcus bacterium, including S.aureus or S.epidermidis. For instance, the immunogen may comprise an IsdA antigen, an IsdB antigen, a CIfA antigen, a ClfB antigen, a SdrD antigen, a Spa antigen, an EsxA antigen, an EsxB antigen, a Sta006 antigen, a hemolysin, and/or a Sta0l 1 antigen. Suitable S.aureus immunogens and 20 their combinations are disclosed in reference 30. The immunogen may elicit an immune response against a meningococcal bacterium (Neisseria meningitidis). For instance, the immunogen may be a capsular saccharide, which may be conjugated to a carrier protein. Capsular saccharides are particularly useful for protecting against meningococcal serogroups A, C, W135 and/or Y. In addition to (or in place of) capsular saccharide immunogen(s), 25 polypeptide immunogens and/or outer membrane vesicles may be used to elicit a protective anti meningococcal immune response, particularly for use against serogroup B e.g. as disclosed in reference 31. Further details of useful serogroup B antigens are given below. The immunogen may elicit an immune response against a hepatitis virus, such as a hepatitis A virus, a hepatitis B virus and/or a hepatitis C virus. For instance, the immunogen may be hepatitis B virus 30 surface antigen (HBsAg). In some embodiments, though, the immunogen is not HBsAg (cf ref. 32). The immunogen may elicit an immune response against a respiratory syncytial virus. Immunogens may be from a group A RSV and/or a group B RSV. Suitable immunogens may comprise the F and/or G glycoproteins or fragments thereof e.g. as disclosed in references 33 & 34. The immunogen may elicit an immune response against a Chlamydia bacterium, including 35 Ctrachornatis and C.pneumoniae. Suitable immunogens include those disclosed in references 35-41. -9- WO 2011/024071 PCT/IB2010/002259 The immunogen may elicit an immune response against an Escherichia coli bacterium, including extraintestinal pathogenic strains. Suitable immunogens include those disclosed in references 42- 45. The immunogen may elicit an immune response against a coronavirus, such as the human SARS coronavirus. Suitable immunogens may comprise the spike glycoprotein. 5 The immunogen may elicit an immune response against a Helicobacter pylori bacterium. Suitable immunogens include CagA [46-49], VacA [50,51], and/or NAP [52-54]. The immunogen may elicit an immune response against rabies virus. A suitable immunogen is an inactivated rabies virus [55, RabAvertTM]. The immunogen may elicit an immune response against a human papillomavirus. Useful 10 immunogens are LI capsid proteins, which can assemble to form structures known as virus-like particles (VLPs). The VLPs can be produced by recombinant expression of Ll in yeast cells (e.g. in S.cerevisiae) or in insect cells (e.g. in Spodoptera cells, such as Sfrugiperda, or in Drosophila cells). For yeast cells, plasmid vectors can carry the LI gene(s); for insect cells, baculovirus vectors can carry the LI gene(s). More preferably, the composition includes LI VLPs from both HPV-16 and 15 HPV- 18 strains. This bivalent combination has been shown to be highly effective [56]. In addition to HPV-16 and HPV-18 strains, it is also possible to include LI VLPs from HPV-6 and HPV-l I strains. The immunogen may elicit an immune response against a tumour antigen, such as MAGE-1, MAGE-2, MAGE-3 (MAGE-A3), MART-1/Melan A, tyrosinase, gplOO, TRP-2, etc. The immunogen may elicit an immunotherapeutic response against lung cancer, melanoma, breast cancer, 20 prostate cancer, etc. The immunogen may elicit an immune response against a hapten conjugated to a carrier protein, where the hapten is a drug of abuse [57]. Examples include, but are not limited to, opiates, marijuana, amphetamines, cocaine, barbituates, glutethimide, methyprylon, chloral hydrate, methaqualone, benzodiazepines, LSD, nicotine, anticholinergic drugs, antipsychotic drugs, tryptamine, other 25 psychomimetic drugs, sedatives, phencyclidine, psilocybine, volatile nitrite, and other drugs inducing physical and/or psychological dependence. Various other immunogens may be used. Serogroup B meningococcus Adjuvants of the invention are useful for enhancing an immune response against meningococcus, and 30 in particular serogroup B meningococcus ("NmB"). Suitable immunogens for eliciting anti-NmB responses include polypeptide antigens, lipooligosaccharide and/or membrane vesicles. Meningococcal volvpeptide antigens A composition may include one or more meningococcal polypeptide antigen(s). For instance, a composition may include a polypeptide antigen selected from the group consisting of: 287, NadA, 35 NspA, HmbR, NhhA, App, Omp85, and/or fHBP. These antigens will usefully be present as purified -10- WO 2011/024071 PCT/IB2010/002259 polypeptides e.g. recombinant polypeptides. The antigen will preferably elicit bactericidal anti meningococcal antibodies after administration to a subject. A composition of the invention may include a 287 antigen. The 287 antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [58] as gene NMB2132 5 (GenBank accession number GI:7227388; SEQ ID NO: 3 herein). The sequences of 287 antigen from many strains have been published since then. For example, allelic forms of 287 can be seen in Figures 5 and 15 of reference 59, and in example 13 and figure 21 of reference 60 (SEQ IDs 3179 to 3184 therein). Various immunogenic fragments of the 287 antigen have also been reported. Preferred 287 antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more 10 identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 3; and/or (b) comprising a fragment of at least 'n consecutive amino acids of SEQ ID NO: 3, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18. 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 3. The most useful 287 antigens of the invention can elicit antibodies which, after 15 administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 3. Advantageous 287 antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. A composition of the invention may include a NadA antigen. The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [58] as gene NN4B1994 20 (GenBank accession number GI:7227256; SEQ ID NO: 4 herein). The sequences of NadA antigen from many strains have been published since then, and the protein's activity as a Neisserial adhesin has been well documented. Various immunogenic fragments of NadA have also been reported. Preferred NadA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 25 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 4; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 4, wherein n' is 7 or more (e.g. 8, 10, 12, 14,16,18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 4. The most useful NadA antigens of the invention can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide 30 consisting of amino acid sequence SEQ ID NO: 4. Advantageous NadA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. SEQ ID NO: 6 is one such fragment. A composition of the invention may include a NspA antigen. The NspA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [58] as gene NMB0663 35 (GenBank accession number GI:7225888; SEQ ID NO: 5 herein). The antigen was previously known from references 61 & 62. The sequences of NspA antigen from many strains have been published since then. Various immunogenic fragments of NspA have also been reported. Preferred NspA -11- WO 2011/024071 PCT/IB2010/002259 antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 5; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 5, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 5 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 5. The most useful NspA antigens of the invention can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 5. Advantageous NspA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 10 Compositions of the invention may include a meningococcal HmbR antigen. The full-length HmbR sequence was included in the published genome sequence for meningococcal serogroup B strain MC58 [58] as gene NMB1668 (SEQ ID NO: 12 herein). The invention can use a polypeptide that comprises a full-length HmbR sequence, but it will often use a polypeptide that comprises a partial HmbR sequence. Thus in some embodiments a HmbR sequence used according to the invention may 15 comprise an amino acid sequence having at least i% sequence identity to SEQ ID NO: 12, where the value of i is 50, 60, 70, 80, 90, 95, 99 or more. In other embodiments a HmbR sequence used according to the invention may comprise a fragment of at least consecutive amino acids from SEQ ID NO: 12, where the value of] is 7, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more. In other embodiments a HmbR sequence used according to the invention may 20 comprise an amino acid sequence (i) having at least i% sequence identity to SEQ ID NO: 12 and/or (ii) comprising a fragment of at least j consecutive amino acids from SEQ ID NO: 12. Preferred fragments of j amino acids comprise an epitope from SEQ ID NO: 12. Such epitopes will usually comprise amino acids that are located on the surface of HmbR. Useful epitopes include those with amino acids involved in HmbR's binding to haemoglobin, as antibodies that bind to these epitopes 25 can block the ability of a bacterium to bind to host haemoglobin. The topology of HmbR, and its critical functional residues, were investigated in reference 63. The most useful HmbR antigens of the invention can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 12. Advantageous HmbR antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a 30 subject. A composition of the invention may include a NhhA antigen. The NhhA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [58] as gene NMB0992 (GenBank accession number GI:7226232; SEQ ID NO: 6 herein). The sequences of NhhA antigen from many strains have been published since e.g. refs 59 & 64, and various immunogenic fragments 35 of NhhA have been reported. It is also known as Hsf. Preferred NhhA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 6; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 6, -12- WO 2011/024071 PCT/IB2010/002259 wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 6. The most useful NhhA antigens of the invention can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 6. 5 Advantageous NhhA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. A composition of the invention may include an App antigen. The App antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [58] as gene NMB1985 (GenBank accession number GI:7227246; SEQ ID NO: 7 herein). The sequences of App antigen 10 from many strains have been published since then. Various immunogenic fragments of App have also been reported. Preferred App antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 7; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 7, wherein 'n' is 7 or more (e.g. 8, 10, 15 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 7. The most useful App antigens of the invention can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 7. Advantageous App antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a 20 subject. A composition of the invention may include an Omp85 antigen. The Omp85 antigen was included in the published genome. sequence for meningococcal serogroup B strain MC58 [58] as gene NMB01 82 (GenBank accession number GI:7225401; SEQ ID NO: 8 herein). The sequences of Omp85 antigen from many strains have been published since then. Further information on Omp85 can be found in 25 references 65 and 66. Various immunogenic fragments of Omp85 have also been reported. Preferred Omp85 antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 8; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 8, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 8. The most useful Omp85 antigens of the invention can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 8. Advantageous Omp85 antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 35 A composition of the invention may include at least one meningococcal factor H binding protein (fHBP). The fHBP antigen has been characterised in detail. It has also been called protein '741' [SEQ IDs 2535 & 2536 in ref. 60], 'NMB1870', 'GNA1870' [refs. 67-69], 'P2086', 'LP2086' or -13- WO 2011/024071 PCT/IB2010/002259 'ORF2086' [70-72]. It is naturally a lipoprotein and is expressed across all meningococcal serogroups. The fHBP antigen falls into three distinct variants [73] and it has been found that serum raised against a given family is bactericidal within the same family, but is not active against strains which express one of the other two families i.e. there is intra-family cross-protection, but not 5 inter-family cross-protection. Compositions of the invention can include a single fHBP variant, but advantageously include fHBP from two or three of the variants [73]. Advantageous fHBP antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. Where a composition comprises a single fHBP variant, it may include one of the following: 10 (a) a first polypeptide comprising a first amino acid sequence, where the first amino acid sequence comprises an amino acid sequence (i) having at least a% sequence identity to SEQ ID NO: 9 and/or (ii) consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 9; (b) a second polypeptide, comprising a second amino acid sequence, where the second amino acid sequence comprises an amino acid sequence (i) having at least b% sequence identity to SEQ ID NO: 15 10 and/or (ii) consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 10; (c) a third polypeptide, comprising a third amino acid sequence, where the third amino acid sequence comprises an amino acid sequence (i) having at least c% sequence identity to SEQ ID NO: II and/or (ii) consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 11. Where a composition comprises two different meningococcal fHBP antigens, it may include a 20 combination of: (i) a first and second polypeptide as defined above; (ii) a first and third polypeptide as defined above; or (iii) a second and third polypeptide as defined above. A combination of a first and third polypeptide is preferred. The first and second polypeptides may be part of a single polypeptide (a fusion protein) comprising the first and second amino acid sequences. Where a composition comprises three different meningococcal fHBP antigens, it may include a 25 combination of: a first and second and third polypeptide as defined above, and these may be part of a single polypeptide (a fusion protein) comprising the first and second and third amino acid sequences. Such triple-fHTBP fusion proteins are disclosed in references 74 and 75 e.g. SEQ ID NO: 17 herein. Where a composition comprises two different meningococcal fl-IBP antigens, although these may share some sequences in common, the first, second and third polypeptides have different fHBP 30 amino acid sequences. A polypeptide comprising the first amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 9 (MC58). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 10 or 35 to the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 11. -14- WO 2011/024071 PCT/IB2010/002259 A polypeptide comprising the second amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 10 (2996). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein having amino acid sequence SEQ ID 5 NO: 9 or to the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 11. A polypeptide comprising the third amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 11 (M1239). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 9 or to 10 the wild-type meningococcus protein having amino acid sequence SEQ ID NO: 10. In some embodiments the fragment of at least x contiguous amino acids from SEQ ID NO: 9 is not also present within SEQ ID NO: 10 or within SEQ ID NO: 11. Similarly, the fragment of at least y contiguous amino acids from SEQ ID NO: 10 might not also be present within SEQ ID NO: 9 or within SEQ ID NO: 11. Similarly, the fragment of at least z contiguous amino acids from SEQ ID 15 NO: 11 might not also be present within SEQ ID NO: 9 or within SEQ ID NO: 10. In some embodiments, when said fragment from one of SEQ ID NOs: 9 to 11 is aligned as a contiguous sequence against the other two SEQ ID NOs, the identity between the fragment and each of the other two SEQ ID NOs is less than 75% e.g. less than 70%, less than 65%, less than 60%, etc. The value of a is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of b 20 is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of c is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The values of a, b and c may be the same or different. In some embodiments, a b and c are identical. The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y 25 is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The values of x, y and z may be the same or different. In some embodiments, xy and z are identical. 30 Fragments preferably comprise an epitope from the respective SEQ ID NO: sequence. Other useful fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the respective SEQ ID NO: while retaining at least one epitope thereof. In some embodiments fHBP polypeptide(s) are lipidated e.g. at a N-terminus cysteine. For lipidated 35 fHlBPs, lipids attached to cysteines will usually include palmitoyl residues e.g. as tripalmitoyl-S -15- WO 2011/024071 PCT/IB2010/002259 glyceryl-cysteine (Pam3Cys), dipalmitoyl-S-glyceryl cysteine (Pam2Cys), N-acetyl (dipalmitoyl-S glyceryl cysteine), etc. A composition may include more than one meningococcal polypeptide antigen. For example, the composition may include three polypeptides: one comprising SEQ ID NO: 13, one comprising SEQ 5 ID NO: 14, and a third comprising SEQ ID NO: 15 or 26 (see references 31 & 76). This combination of three polypeptides, covering five antigens, is particularly advantageous for protecting against serogroup B meningococcus. An alternative composition may include: a first polypeptide comprising SEQ ID NO: 13, a second polypeptide comprising SEQ ID NO: 15, and a third comprising SEQ ID NO: 17. An alternative composition may include: a first polypeptide comprising SEQ ID NO: 13, a 10 second polypeptide comprising SEQ ID NO: 15, and a third comprising SEQ ID NO: 18. An alternative composition may include: a first polypeptide comprising SEQ ID NO: 13, a second polypeptide comprising SEQ ID NO: 26, and a third comprising SEQ ID NO: 17. An alternative composition may include: a first polypeptide comprising SEQ ID NO: 13, a second polypeptide comprising SEQ ID NO: 26, and a third comprising SEQ ID NO: 18. 15 Meningococcal lipooligosaccharide A composition may include one or more meningococcal lipooligosaccharide (LOS) antigen(s). Meningococcal LOS is a glucosamine-based phospholipid that is found in the outer monolayer of the outer membrane of the bacterium. It includes a lipid A portion and a core oligosaccharide region, with the lipid A portion acting as a hydrophobic anchor in the membrane. Heterogeneity within the 20 oligosaccharide core generates structural and antigenic diversity among different meningococcal strains, which has been used to subdivide the strains into 12 immunotypes (LI to L12). The invention may use LOS from any immunotype e.g. from Li, L2, L3, L4, L5, L6, L7 and/or L8. The L2 and L3 a-chains naturally include lacto-N-neotetraose (LNnT). Where the invention uses LOS from a L2 or L3 immunotype this LNnT may be absent. This absence can be achieved 25 conveniently by using mutant strains that are engineered to disrupt their ability to synthesise the LNnT tetrasaccharide within the a-chain. It is known to achieve this goal by knockout of the enzymes that are responsible for the relevant biosynthetic additions [77,107]. For instance, knockout of the LgtB enzyme prevents addition of the terminal galactose of LNnT, as well as preventing downstream addition of the a-chain's terminal sialic acid. Knockout of the LgtA enzyme prevents 30 addition of the N-acetyl-glucosamine of LNnT, and also the downstream additions. LgtA knockout may be accompanied by LgtC knockout. Similarly, knockout of the LgtE and/or GalE enzyme prevents addition of internal galactose, and knockout of LgtF prevents addition of glucose to the Hepl residue. Any of these knockouts can be used, singly or in combination, to disrupt the LNnT tetrasaccharide in a L2, L3, L4, L7 or L9 immunotype strain. Knockout of at least LgtB is preferred, 35 as this provides a LOS that retains useful immunogenicity while removing the LNnT epitope. In addition to, or in place of, mutations to disrupt the LNnT epitope, a knockout of the galE gene also provides a useful modified LOS, and a lipid A fatty transferase gene may similarly be knocked out -16- WO 2011/024071 PCT/IB2010/002259 [78]. At least one primary O-linked fatty acid may be removed from LOS [79]. LOS having a reduced number of secondary acyl chains per LOS molecule can also be used [80].The LOS will typically include at least the GIcNAc-Hep 2 phosphoethanolamine-KDO 2 -Lipid A structure [81]. The LOS may include a GlcNAcP 1 -3Galp -4Glc trisaccharide while lacking the LNnT tetrasaccharide. 5 LOS may be included in various forms. It may be used in purified form on its own. It may be conjugated to a carrier protein. When LOS is conjugated, conjugation may be via a lipid A portion in the LOS or by any other suitable moiety e.g. its KDO residues. If the lipid A moiety of LOS is absent then such alternative linking is required. Conjugation techniques for LOS are known from e.g. references 79, 81, 82, 83, etc. Useful carrier proteins for these conjugates include e.g. bacterial 10 toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. The LOS may be from a strain (e.g. a genetically-engineered meningococcal strain) which has a fixed (i.e. not phase variable) LOS immunotype as described in reference 84. For example, L2 and L3 LOS immunotypes may be fixed. Such strains may have a rate of switching between immunotypes that is reduced by more than 2-fold (even >50_fold) relative to the original wild-type 15 strain. Reference 84 discloses how this result can be achieved by modification of the lgtA and/or IgtG gene products. LOS may be O-acetylated on a GlcNac residue attached to its Heptose II residue e.g. for L3 [85]. An immunogenic composition can include more than one type of LOS e.g. LOS from meningococcal immunotypes L2 and L3. For example, the LOS combinations disclosed in reference 86 may be used. 20 A LOS antigen can preferably elicit bactericidal anti-meningococcal antibodies after administration to a subject. Membrane vesicles A composition may include meningococcal outer membrane vesicles. These include any proteoliposomic vesicle obtained by disruption of or blebbling from a meningococcal outer 25 membrane to form vesicles therefrom that include protein components of the outer membrane. Thus the term includes OMVs (sometimes referred to as 'blebs'), microvesicles (MVs [87]) and 'native OMVs' ('NOMVs' [88]). MVs and NOMVs are naturally-occurring membrane vesicles that form spontaneously during bacterial growth and are released into culture medium. MVs can be obtained by culturing Neisseria 30 in broth culture medium, separating whole cells from the smaller MVs in the broth culture medium (e.g. by filtration or by low-speed centrifugation to pellet only the cells and not the smaller vesicles), and then collecting the MVs from the cell-depleted medium (e.g. by filtration, by differential precipitation or aggregation of MVs, by high-speed centrifugation to pellet the MVs). Strains for use in production of MVs can generally be selected on the basis of the amount of MVs produced in 35 culture e.g. refs. 89 & 90 describe Neisseria with high MV production. -17- WO 2011/024071 PCT/IB2010/002259 OMVs are prepared artificially from bacteria, and may be prepared using detergent treatment (e.g. with deoxycholate), or by non-detergent means (e.g. see reference 91). Techniques for forming OMVs include treating bacteria with a bile acid salt detergent (e.g. salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., 5 with sodium deoxycholate [92 & 93] being preferred for treating Neisseria) at a pH sufficiently high not to precipitate the detergent [94]. Other techniques may be performed substantially in the absence of detergent [91] using techniques such as sonication, homogenisation, microfluidisation, cavitation, osmotic shock, grinding, French press, blending, etc. Methods using no or low detergent can retain useful antigens such as NspA [91]. Thus a method may use an OMV extraction buffer with about 10 0.5% deoxycholate or lower e.g. about 0.2%, about 0.1%, <0.05% or zero. A useful process for OMV preparation is described in reference 95 and involves ultrafiltration on crude OMVs, rather than instead of high speed centrifugation. The process may involve a step of ultracentrifugation after the ultrafiltration takes place. Vesicles for use with the invention can be prepared from any meningococcal strain. The vesicles will 15 usually be from a serogroup B strain, but it is possible to prepare them from serogroups other than B (e.g. reference 94 discloses a process for serogroup A), such as A, C, WI 35 or Y. The strain may be of any serotype (e.g. 1, 2a, 2b, 4, 14, 15, 16, etc.), any serosubtype, and any immunotype (e.g. Ll; L2; L3; L3,3,7; LI0; etc.). The meningococci may be from any suitable lineage, including hyperinvasive and hypervirulent lineages e.g. any of the following seven hypervirulent lineages: 20 subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37 complex; A4 cluster; lineage 3. These lineages have been defined by multilocus enzyme electrophoresis (MLEE), but multilocus sequence typing (MLST) has also been used to classify meningococci [ref. 96] e.g. the ET-37 complex is the ST-Il complex by MLST, the ET-5 complex is ST-32 (ET-5), lineage 3 is ST-41/44, etc. Vesicles can be prepared from strains having one of the following subtypes: P1.2; P1.2,5; P1.4; 25 P1.5; P1.5,2; PI.5,c; P1.5c,10; P1.7,16; PI.7,16b; Pl.7h,4; P1.9; P1.15; P1.9,15; P1.12,13; P1.13; P1.14; P1.21,16; P1.22,14. Vesicles used with the invention may be prepared from wild-type meningococcal strains or from mutant meningococcal strains. For instance, reference 97 discloses preparations of vesicles obtained from N.meningitidis with a modifiedfur gene. Reference 105 teaches that nspA expression should be 30 up-regulated with concomitant porA and cps knockout. Further knockout mutants of N.meningitidis for OMV production are disclosed in references 105 to 107. Reference 98 discloses vesicles in which fHBP is upregulated. Reference 99 discloses the construction of vesicles from strains modified to express six different PorA subtypes. Mutant Neisseria with low endotoxin levels, achieved by knockout of enzymes involved in LPS biosynthesis, may also be used [100,101]. These or others 35 mutants can all be used with the invention. Thus a strain used with the invention may in some embodiments express more than one PorA subtype. 6-valent and 9-valent PorA strains have previously been constructed. The strain may -18- WO 2011/024071 PCT/IB2010/002259 express 2, 3, 4, 5, 6, 7, 8 or 9 of PorA subtypes: P1.7,16; P1.5-1,2-2; P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1 and/or P1.1 8-1,3,6. In other embodiments a strain may have been down-regulated for PorA expression e.g. in which the amount of PorA has been reduced by at least 20% (e.g. >30%, >40%, >50%, >60%, a70%, >80%, >90%, >95%, etc.), or even knocked 5 out, relative to wild-type levels (e.g. relative to strain H44/76, as disclosed in reference 108). In some embodiments a strain may hyper-express (relative to the corresponding wild-type strain) certain proteins. For instance, strains may hyper-express NspA, protein 287 [102], fHBP [98], TbpA and/or TbpB [103], Cu,Zn-superoxide dismutase [103], HmbR, etc. In some embodiments a strain may include one or more of the knockout and/or hyper-expression 10 mutations disclosed in references 104 to 107. Preferred genes for down-regulation and/or knockout include: (a) Cps, CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [104]; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA, and/or ThpB [105]; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, 15 PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [106]; and (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PiIC, PorB, SiaD, SynA, SynB, and/or SynC [107]. Where a mutant strain is used, in some embodiments it may have one or more, or all, of the following characteristics: (i) down-regulated or knocked-out LgtB and/or GalE to truncate the meningococcal LOS; (ii) up-regulated ThpA; (iii) up-regulated NhhA; (iv) up-regulated Omp85; (v) up-regulated 20 LbpA; (vi) up-regulated NspA; (vii) knocked-out PorA; (viii) down-regulated or knocked-out FrpB; (ix) down-regulated or knocked-out Opa; (x) down-regulated or knocked-out Opc; (xii) deleted cps gene complex. A truncated LOS can be one that does not include a sialyl-lacto-N-neotetraose epitope e.g. it might be a galactose-deficient LOS. The LOS may have no a chain. If LOS is present in a vesicle it is possible to treat the vesicle so as to link its LOS and protein 25 components ("intra-bleb" conjugation [107]). The invention may be used with mixtures of vesicles from different strains. For instance, reference 108 discloses vaccine comprising multivalent meningococcal vesicle compositions, comprising a first vesicle derived from a meningococcal strain with a serosubtype prevalent in a country of use, and a second vesicle derived from a strain that need not have a serosubtype prevent in a country of 30 use. Reference 109 also discloses useful combinations of different vesicles. A combination of vesicles from strains in each of the L2 and L3 immunotypes may be used in some embodiments. Adsorption Aluminium salt adjuvants are adsorptive i.e. immunogens can adsorb to the salts, by a variety of mechanisms. In some circumstances, however, immunogen and aluminium salt adjuvants can both be 35 present in a composition without adsorption, either through an intrinsic property of the immunogen -19- WO 2011/024071 PCT/IB2010/002259 or because of steps taken during formulation (e.g. the use of an appropriate pH during formulation to prevent adsorption from occurring). Preferred complexes of immunostimulatory oligonucleotide and polycationic polymer are also adsorptive. 5 In a mixture of aluminium salts and complexes, therefore, there can be multiple adsorptive opportunities for an immunogen: an immunogen can adsorb to aluminium salt, to a oligonucleotide/polymer complex, to both (in various proportions), or to neither. The invention covers all such arrangements. For example, in one embodiment an immunogen can be adsorbed to an aluminium salt, and the adsorbed immunogen/salt can then be mixed with an 10 oligonucleotide/polymer complex. In another embodiment an immunogen can be adsorbed to an oligonucleotide/polymer complex, and the adsorbed immunogen/complex can then be mixed with an. aluminium salt. In another embodiment two immunogens (the same or different) can be separately adsorbed to an oligonucleotide/polymer complex and to an aluminium salt, and the two adsorbed components can then be mixed. 15 In some situations, an immunogen may change its adsorption status e.g. by a change in pH or temperature, or after mixing of components. Desorption of antigens from aluminium salts in vitro [110] and in vivo [111] is known. Desorption from one adsorptive particle followed by resorption to a different adsorptive particle can occur, thereby resulting in e.g. transfer of an immunogen from an aluminium salt adjuvant to a complex or vice versa. In some embodiments, a single antigen molecule 20 or complex might adsorb to both an aluminium salt and a complex, forming a bridge between the two adsorptive particles. If an inmunogen adsorbs to an adsorptive component, it is not necessary for all of the immunogen to adsorb. This situation can occur because of an immunogen's intrinsic equilibrium between adsorbed and soluble phases, or because adsorptive surfaces are saturated. Thus the immunogen in a 25 composition may be fully or partially adsorbed, and the adsorbed fraction can be on one or more different adsorptive components (e.g. on aluminium salt and/or on a oligonucleotide/polymer. complex). In this situation, the adsorbed fraction may be at least 10% (by weight) of the total amount of that immunogen in the composition e.g. >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >98% or more. In some embodiments an immunogen is totally adsorbed i.e. none is 30 detectable in the supernatant after centrifugation to separate complexes from bulk liquid medium. In other embodiments, though, none of a particular immunogen may be adsorbed. In some circumstances it is possible that the immunostimulatory oligonucleotide and/or polycationic polymer component of a complex could adsorb to the aluminium salt. Preferably, though, the complexes remain intact after mixing with the aluminium salt. Also, to avoid adsorption of 35 complexes to the aluminium salt (and vice versa) it is useful that the aluminium salt and the complexes have similar points of zero charge (isoelectric points) e.g. within 1 pH unit of each other. -20- WO 2011/024071 PCT/IB2010/002259 Thus useful complexes have a PZC of between 10 and 12, which is useful for combining with an aluminium hydroxide adjuvant having a PZC of about 11. Packaging of compositions or kit components Suitable containers for adjuvant compositions, immunogenic compositions and kit components of the 5 invention include vials, syringes (e.g. disposable syringes), etc. These containers should be sterile. The containers can be packaged together to form a kit e.g. in the same box. Where a component is located in a vial, the vial can be made of a glass or plastic material. The vial is preferably sterilized before the composition is added to it. To avoid problems with latex-sensitive subjects, vials are preferably sealed with a latex-free stopper, and the absence of latex in all 10 packaging material is preferred. The vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial) e.g. 10 doses. Useful vials are made of colorless glass. Borosilicate glasses are preferred to soda lime glasses. Vials may have stoppers made of butyl rubber. A vial can have a cap (e.g. a Luer lock) adapted such that a syringe can be inserted into the cap. A vial cap may be located inside a seal or cover, such that the seal or cover has to be removed before 15 the cap can be accessed. A vial may have a cap that permits aseptic removal of its contents, particularly for multidose vials. Where a component is packaged into a syringe, the syringe may have a needle attached to it. If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed. The plunger in a syringe may have a stopper to prevent the plunger from 20 being accidentally removed during aspiration. The syringe may have a latex rubber cap and/or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap may be made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield. Useful syringes are those marketed under the trade name "Tip-Lok"TM. 25 Containers may be marked to show a half-dose volume e.g. to facilitate delivery to children. For instance, a syringe containing a 0.5ml dose may have a mark showing a 0.25ml volume. It is usual in multi-component products to include more material than is needed for subject administration, so that a full final dose volume is obtained despite any inefficiency in material transfer. Thus an individual container may include overfill e.g. of 5-20% by volume. 30 Methods of treatment, and administration of immunogenic compositions Compositions of the invention are suitable for administration to human subjects, and the invention provides a method of raising an immune response in a subject, comprising the step of administering an immunogenic composition of the invention to the subject. -21- WO 2011/024071 PCT/IB2010/002259 The invention also provides a method of raising an immune response in a subject, comprising the step of mixing the contents of the containers of a kit of the invention and administering the mixed contents to the subject. The invention also provides composition or kit of the invention for use as a medicament e.g. for use 5 in raising an immune response in a subject. The invention also provides the use of an aluminium salt, an immunostimulatory oligonucleotide and a polycationic polymer, in the manufacture of a medicament for raising an immune response in a subject. This medicament may be administered in combination with an immunogen. The invention also provides the use of an aluminium salt, an immunostimulatory oligonucleotide, a 10 polycationic polymer and an immunogen, in the manufacture of a medicament for raising an immune response in a subject. These methods and uses will generally be used to generate an antibody response, preferably a protective antibody response. Compositions of the invention can be administered in various ways. The usual immunisation route is 15 by intramuscular injection (e.g. into the arm or leg), but other available routes include subcutaneous injection, intranasal, oral, buccal, sublingual, intradermal, transcutaneous, transdermal, etc. Immunogenic compositions prepared according to the invention may be used as vaccines to treat both children and adults. A subject may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Subjects for receiving the vaccines may be elderly (e.g. >50 years 20 old, >60 years old, and preferably >65 years), the young (e.g. :5 years old), hospitalised subjects, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, immunodeficient subjects, people travelling abroad, etc. Aluminium salt adjuvants are routinely used in infant populations, and IC31 TM has also been effective in this age group [22,112]. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population. 25 Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive subjects. Multiple doses will 30 typically be administered at least I week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.). General The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. -22- WO 2011/024071 PCT/IB2010/002259 The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention. The term "about" in relation to a numerical value x is optional and means, for example, x+10%. 5 Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc. Where animal (and particularly bovine) materials are used in the culture of cells, they should be 10 obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal-derived materials. Where a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug. 15 Where a cell substrate is used for reassortment or reverse genetics procedures, or for viral growth, it is preferably one that has been approved for use in human vaccine production e.g. as in Ph Eur general chapter 5.2.3. In some embodiments, the invention does not encompass or use compositions which include an immunogen comprising amino acid sequence SEQ ID NO: 16 [113]. In embodiments where an 20 immunogen comprising amino acid sequence SEQ ID NO: 16 is present in a composition, the aluminium salt will typically be aluminium hydroxide and/or the composition may include at least one further and different meningococcal immunogen. MODES FOR CARRYING OUT THE INVENTION Adjuvants 25 An aluminium hydroxide adjuvant suspension is prepared by standard methods. IC31 complexes were prepared as disclosed in reference 19. Adjuvant combinations were made my mixing the aluminium hydroxide adjuvant with IC31 complexes. The individual adjuvants (Al-H, IC3 1) and their mixture (Al-H+1C3 1) have been combined with antigens from various pathogens and tested in various animal models. Various orders of mixing Al-H, IC31 and antigen have been used. 30 Antigens tested so far are from pathogens including N.meningitidis serogroups A/B/C/W135/-Y, extraintestinal pathogenic E.coli, Streptococcus pyogenes, Candida albicans, respiratory syncytial virus and S.aureus. These studies have mainly used polypeptide antigens, but also used saccharide antigens (meningococcal serogroups A, C, W 135 & Y, and C.albicans). -23- WO 2011/024071 PCT/IB2010/002259 Meningococcus (i) The three polypeptides which make up the '5CVMB' vaccine disclosed in reference 31 were adjuvanted with aluminium hydroxide and/or IC31. The encoded polypeptides have amino acid sequences SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 (see refs. 31 & 76). 5 In a first set of experiments, nine groups of mice received lOpg of antigens, 3mg/mi of aluminium hydroxide and varying doses of IC3 1. Groups received the following nine compositions, with groups 7-9 receiving the same antigens as 1-6 but differently formulated: Antigen dose (g) IC31 volume* (pl) Al-H (mg/ml) 1 10 100 3 2 10 50 3 3 10 25 3 4 10 10 3 5 10 0 3 6 10 100 0 7 10 0 3 8 10 100 3 9 10 100 0 * A standard IC31 suspension was used. 100pl of this suspension gave full-strength. Lower volumes gave lower strengths. To preserve the volume for the lower-strength compositions, buffer was added up to 1001. 10 Sera from the mice were tested against a panel of meningococcal strains for bactericidal activity. Bactericidal titers from experiment MPO3 were as follows against six different strains, A to F: A B C D E F 1 >65536 4096 8192 4096 256 32768 2 >65536 8192 8192 8192 512 >65536 3 >65536 4096 4096 8192 512 32768 4 >65536 2048 4096 4096 512 8192 5 >65536 2048 4096 8192 256 32768 6 >65536 4096 >8192 8192 1024 >65536 7 >65536 2048 4096 4096 256 4096 8 >65536 >8192 >8192 >8192 512 >65536 9 32768 8192 >8192 >8192 4096 >65536 Thus the titers obtained with Al-H as the only adjuvant (group 5) were generally improved across the panel by the addition of IC31 at various ratios (groups 1 to 4). The same effect was seen with the 15 different antigen formulation (compare groups 7 and 8) Further studies on a wider panel confirmed that addition of IC31 improved the titers when compared to the use of Al-H alone. For instance, bactericidal titers using sera from groups 1, 3 and 5 against a panel of 16 strains (including A to F from above) were as follows, with 50% of strains in group 5 having a titer >1:1024 but 56.25% in groups 1 and 3: -24- WO 2011/024071 PCT/IB2010/002259 1 3 5 Al-H + IC31(100) Al-H + IC31(25) Al-H A >65536 >65536 >65536 B 4096 4096 2048 C 8192 4096 4096 D 4096 8192 8192 E 256 512 256 F 32768 32768 32768 G 4096 2048 2048 H 2048 2048 1024 I 64 256 64 J 512 256 128 K 256 256 256 L 512 512 512 M 2048 512 1024 N 1024 2048 512 0 128 64 64 P 512 1024 512 The nine compositions were tested for pH and osmolality. For compositions 1-5, 7 and 8 the pH was in the range of 6.2 to 6.6; compositions 6 and 9 had a slightly higher pH, in the range 6.9 to 7.3. Osmolality of all compositions was in the range of 280-330 mOsm/kg. 5 SDS-PAGE analysis of the supernatants of the compositions, and of compositions treated to desorb antigens, showed that the antigens adsorbed similarly to the mixture of Al-H+IC31 as to Al-H alone: for all compositions containing Al-H, between 94-100% of each antigen was adsorbed. Meningococcus (ii) A triple-fusion polypeptide containing three variants of fHBP, in the order II-III-I (as disclosed in 10 reference 17; SEQ ID NO: 17 herein), was adjuvanted with aluminium hydroxide and/or IC3 1. In a first set of experiments, six groups of mice received 20 ig of antigen (with or without a purification tag), 3mg/mI of aluminium hydroxide and 1 00pl of IC3 1. Groups received the following: Antigen dose (ptg) Antigen tag IC31 volume (I) Al-H (mg/ml) 1 20 No 100 0 2 20 Yes 100 0 3 20 No 100 3 4 20 Yes 100 3 5 20 No 0 3 6 20 Yes 0 3 Sera from the mice were tested against a panel of meningococcal strains for bactericidal activity. -25- WO 2011/024071 PCT/IB2010/002259 Bactericidal titers from experiment MP05 were again tested against a panel of strains (25 in total). 64% of strains in group 5 (Al-H, no tag) had a titer >1:128, and 36% had a titer >1: 1024, but addition of IC31 (group 3) increased these figures to 76% and 56%, respectively. Similarly, 76% of strains in group 6 (Al-H, tag) had a titer >1: 128, and 64% had a titer >1:1024, but addition of IC31 (group 4) 5 increased these figures to 80% and 68%, respectively. For 23/25 strains the titers were the same or better in group 3 than in group 5; for 22/25 strains the titers were the same or better in group 4 than in group 6. Particularly good improvements were seen against strains NMOO8 and M4287, where very low titers of <1:32 were improved to between 1:256 and 1:4096. The tag-free compositions (1, 3 and 5) were tested for pH and osmolality. The pH was in the range of 10 6.87 to 7.00. Osmolality was in the range of 302-308 mOsm/kg. SDS-PAGE analysis of the supernatants of the tag-free compositions (1, 3 & 5), and of compositions treated to desorb the antigen, showed that the antigen adsorbed similarly to the mixture of Al-H+IC31 as to Al-H alone. Further immunogenicity experiments used the fHBP 1

.

1 1 i-I antigen in combination with the NadA and 15 287-953 antigens (SEQ ID NOs: 13 and 15) in experiment MPO4, with the same groupings and strain panel. Titers in group 4 were the same or better than in group 6 for 24/25 strains; titers were the same or better in group 3 than in group 5 also for 24/25 strains. The proportion of strains where the sera had a bactericidal titer of at least 1:128 was 100% in each of groups I to 4 and in group 6, but only 84% in group 5. With a more stringent threshold of >1:1024, however, sera from groups I to 4 were 20 bactericidal against 88% of strains, whereas the proportion was only 80% in group 6 and 56% in group 5. Thus the anti-meningococcus immune responses obtained with Al-H as the only adjuvant were generally improved across the panel by the addition of IC3 1. Meningococcus (iii) Reference 113 discloses various modified forms of fHBP, including: 25 PATCH_2S (SEQ ID NO: 19 herein) PATCH_5bis (SEQ ID NO: 20 herein) PATCH_5penta (SEQ ID NO: 21 herein) PATCH_9C (SEQ ID NO: 16 herein) PATCHOA (SEQ ID NO: 22 herein) 30 These five modified fHBP proteins, and also PATCH_9F and the wild-type sequence from strain 2996, were used to immunise mice with aluminium hydroxide (Al-H), Al-H+IC3 1, or IC31 alone. Sera were tested against a panel of ten different meningococcal strains. The titers against all 10 strains were as good as or higher in the Al-H+IC31 group than in both the Al-H group and the IC31 group for the PATCH_5B and PATCH_9C sequences. 35 More generally, the invention can be used with a protein comprising any one of SEQ ID NOs: 1 to 80 from reference 113. -26- WO 2011/024071 PCT/IB2010/002259 The combination of Al-H+IC31 also gave better results than Al-H alone when tested with fusion proteins containing PATCH_9C and/or PATCH_10A and/or the wild-type MC58 fHBP sequence (comprising SEQ ID NO: 3) and/or 936 antigen (comprising SEQ ID NO: 23) e.g. converting efficacy against only 1/10 strains with Al-H into efficacy against 9/10 strains when using a fusion 5 protein containing 936 fused to two copies of PATCH_10A (SEQ ID NO: 18). Using the 936-IOA-1OA sequence or a 936-9C-IOA sequence (SEQ ID NO: 25), bactericidal titers against a panel of 10 strains were: 936-10A-IOA 936-9C-10A Al-H Al-H + IC31 Al-H Al-H + IC31 A 8192 >32768 8192 >32768 B <16 4096 128 2048 C 512 8192 1024 >8192 D 256 2048 512 4096 E 16 1024 256 4096 F 64 8192 2048 16384 G 128 2048 1024 4096 HI 16 1024 1024 4096 I 64 4096 128 2048 J _64 J32 256 1024 With one exception, therefore, the addition of IC31 improved titers. 10 The 936-I0A-10A and 936-9C-10A polypeptides were confirmed to be fully adsorbed in the Al-H+IC31 formulations. In further work the 936-1 OA- I 0A polypeptide was formulated with Al-H in a composition including 9mg/ml NaCl and 10mM histidine, pH 6.5. Water for injection and histidine buffer were mixed, and then NaCl was added to give a final osmolality of 308 mOsm/kg. Al-H was added to give a final 15 Al.. concentration of 3mg/ml. The polypeptide was added to give a final concentration of 1 00pg/ml, left for 15 minutes under stirring at room temperature, and then stored overnight at 4'C. Just before administration, IC31 (with a 25:1 molar ratio of peptide:DNA, Ipmol peptide) was added as an aqueous suspension, mixing equal volumes. The final mixture was isotonic and at physiological pH. Polypeptide adsorption was >90% (similar to the level seen with the Al-H alone). 20 Animals (6-week-old CD1 female mice), 8 per group, received 20pg adjuvanted polypeptide intraperitoneally at day 0, with booster doses at days 21 & 35. Blood samples for analysis were taken on day 49 and were analysed by bactericidal assay, in the presence of rabbit or human complement, against a panel of I1 meningococcal strains. Titers were as follows: Rabbit complement Human complement A 16384 1024 -27- WO 2011/024071 PCT/IB2010/002259 B 2048 512 C 4096 >512 D 4096 256 E 4096 256 F 8192 64 G 4096 256 H 1024* 256 I 4096 J 1024 256 K - 512 * = bacteriostatic titer Similar results were seen whether the 936-1OA-10A polypeptide did or did not have a C-terminus hexahistidine tag (SEQ ID NOs: 18 vs. 24). The 936-1OA-10A polypeptide was substituted for the '936-741' polypeptide from 5CVMB, to give 5 a mixture of three polypeptides having amino acid sequences of SEQ ID NOs: 13, 15 and 18/24. Bactericidal titers were similar, with a slight superiority when using the polypeptide without a histidine tag. Compared to Al-H alone, IC31 alone, or Mff59, the Al-H+IC31 mixture gave the best bactericidal strain coverage when using the 936-1 OA- I OA polypeptide, either alone or in combination with other 10 polypeptides from the 5CVMB mixture. Meningococcus (iv) The three polypeptides which make up the '5CVMB' vaccine disclosed in reference 31 were combined with a tetravalent mixture of meningococcal conjugates against serogroups A, C, W135 and Y. The mixture was adjuvanted with Al-H and/or IC31 (at high or low concentration). 15 Bactericidal titers were as follows against a panel with one strain from each of serogroups A, C, W135 and Y: A C W135 Y Un-immunised <16 <16 <16 <16 No adjuvant 1024 256 128 512 IC31high 32768 16384 4096 4096 IC31'*w 16384 8192 1024 2048 Al-hydroxide 16384 8192 1024 4096 Al-H+IC31"gh 16384 32768 4096 8192 Al-H+IC31'ow 8192 65536 2048 8192 Thus the best titers against serogroups C, W135 and Y were seen when using a combination of aluminium hydroxide with IC3 1. -28- WO 2011/024071 PCT/IB2010/002259 E.coli The AcfD protein of E.coli (originally disclosed as SEQ ID NO: 3526 in reference 45; see also reference 44) is a useful immunogen. This antigen has been used to immunise mice in combination with various adjuvants, either alone or in combination. The immunised mice are then challenged with 5 a lethal dose of E.coli and survival is assessed. Survival in control groups ranged from 0-25%, whereas survival after immunisation/challenge with adjuvanted AcfD was as follows: Adjuvant Survival (10% ranges) Freund's complete 80-90% Al-H 70-80% MF59 70-80% IC31 70-80% MF59 + IC31 80-90% Al-H + IC31 100% Thus the best results were seen with the combination of Al-H and IC3 1. Protection correlated with the reduction in bacterial load in the blood of infected animals. 10 It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. REFERENCES [1] Vaccine Design: The Subunit and Adjuvant Approach (Powell & Newman) 1995 (ISBN 0-306-44867-X). [2] Clausi et al. (2008) JPharm Sci DOI 10.1002/jps.21390 [3] Krieg (2003) Nature Medicine 9:831-835. [4] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32:179-185. [5] W098/40100. [6] US 6,207,646. [7] US 6,239,116. [8] US 6,429,199. [9] WOO1/93905. [10] W002/32451. [11] Aichinger et al. (2008) Cell Biology International 32:1449-58. [12] Alvarez-Bravo et al. (1994) Biochem J302:535-8. [13] Nakajima etal. (1997) FEBSLetts 415:64-66. [14] Fritz et al. (2004) Vaccine 22:3274-84. [15] Schellack et al. (2006) Vaccine 24:5461-72. [ 16] Lingnau et al. (2007) Expert Rev Vaccines 6:741-6. [17] W02004/084938. [18] Kamath etal. (2008) EurJlmmunol 38:1247-56. [19] Riedl et al. (2008) Vaccine 26:3461-8. [20] Kritsch et al. (2005) J Chromatography B 822:263-70. -29- WO 2011/024071 PCT/IB2010/002259 (21] Lingnau et aL. (2003) Vaccine 20:3498-508. [22] Olafsdottir et al. (2009) Scand JImmunol 69:194-202. [23] Hem & HogenEsch (2007) Expert Rev Vaccines 6:685-98. [24] Gennaro (2000) Remington: The Science and Practice ofPharmacy. 20th edition, ISBN: 0683306472. [25] Treanor et al. (1996) JInfect Dis 173:1467-70. [26] Keitel et aL. (1996) Clin Diagn Lab Immunol 3:507-10. [27] W003/097091. [28] Cassone & Torosantucci (2006) Expert Rev Vaccines 5:859-67. [29] PCT/IB2010/052445. [30] PCT/IB2010/000998. [31] Giuliani et al. (2006) Proc NatlAcad Sci USA. 103:10834-9. [32] W02004/084937. [33] W095/27787. [34] W003/010317. [35] W02007/110700. [36] W02006/138004. [37] W02005/084306. [38] W02005/002619. [39] W003/049762. [40] W002/02606. [41] WOOO/37494. [42] W02008/020330. [43] W02006/091517. [44] WO/2009/104092. [45] W02006/089264. [46] Covacci & Rappuoli (2000) J. Exp. Med. 19:587-592. [47] WO 93/18150. [48] Covacci et al. (1993) Proc. Nat?. Acad. Sci. USA 90:5791-5795. [49] Tummuru et al. (1994) Infect. Immun. 61:1799-1809. [50] Marchetti et al. (1998) Vaccine 16:33-37. [51] Telford et al. (1994) J. Exp. Med. 179:1653-1658. [52] Evans et al. 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[81] US-6645503 [82] W003/070282. [83] W094/08021 [84] W02004/015099. [85] W02007/144316. [86] W02007/144317. [87] W002/09643. [88] Katial et al. (2002) Infect. Immun. 70:702-707. [89] US 6,180,111. [90] WO01/34642. [91] W02004/019977. [92] EP-00 11243. [93] Fredriksen et al. (1991) NIPHAnn. 14(2):67-80. [94] WO01/91788. [95] W02005/004908. [96] Maiden et aL. (1998) PNAS USA 95:3140-3145. [97] W098/56901. [98] W02006/081259. [99] Claassen et al. (1996) 14(10):1001-8. [100] W099/10497. [101] Steeghs et al. (2001) The EMBO Journal 20:6937-6945. [102] W001/52885. [103] W00/25811. [104] W001/09350. [105] W002/09746. [106] W002/062378. [107] W02004/014417. [108] W003/105890. [109] W02006/024946 [110] W02009/081172. [111] Seeber et al. (1991) JParenteral Sci Technol 45:156-9. [112] Kamath et al. (2008) PLoS ONE 3(11):e3683. [113] W02009/104097. -31-

Claims (21)

1. An immunogenic composition comprising (i) an immunological adjuvant comprising an aluminium salt, an immunostimulatory oligonucleotide and a polycationic polymer, wherein the oligonucleotide and the polymer are associated with each other to form a complex and wherein the polycationic polymer is a peptide which includes one or more Lys-Leu dipeptide sequence(s) and/or one or more Lys-Leu-Lys tripeptide sequence(s); and (ii) an immunogen which elicits an immune response that protects against infection by a bacterial or a fungal agent of disease.

2. A kit when used to prepare an immunogenic composition according to claim 1, said kit comprising: (i) a first container that contains adjuvant of claim 1; and (ii) a second container that contains the immunogen which elicits a protective immune response against infection by a bacterial or fungal agent of disease and optionally, a further adjuvant.

3. The immunogenic composition according to claim 1 or the kit according to claim 2, wherein the aluminium salt is an aluminium hydroxide.

4. The immunogenic composition according to claim 1 or the kit according to claim 2, wherein the aluminium salt is an aluminium phosphate.

5. The immunogenic composition according to any one of claims I to 4 or the kit according to any one of claims 2 to 4, wherein the oligonucleotide is single-stranded and has between 10 and 100 nucleotides.

6. The immunogenic composition or kit according to claim 5, wherein the oligonucleotide is 5' (IC)13-3'.

7. The immunogenic composition according to any of claims 1 to 6 or the kit according to any one of claims 2 to 6, wherein the peptide has between 5 and 50 amino acids.

8. The immunogenic composition or kit according to claim 7, wherein the peptide has the amino acid sequence KLKLLLLLKLK.

9. The immunogenic composition according to any one of claims 1 to 8 or the kit according to any one of claims 2 to 8, wherein the oligonucleotide and polymer are present at a molar ratio 1:25.

10. The immunogenic composition according to any one of claims 1 to 9 or the kit according to any one of claims 2 to 9, wherein the aluminium salt and the complex are both particulate with a mean particle diameter between 1-20um. - 32 -

11. The immunogenic composition according to any one of claims I to 10 or the kit according to any one of claims 2 to 10, wherein the adjuvant is sterile.

12. The immunogenic composition or kit according to claim 11, wherein the sterile adjuvant is prepared by a process comprising: (i) filter sterilising an immunostimulatory oligonucleotide and a polycationic polymer; (ii) mixing the filter sterilised immunostimulatory oligonucleotide and a polycationic polymer under sterile conditions to form sterile complexes; and (iii) mixing the sterile complexes with a sterile aluminium salt.

13. The immunogenic composition according to any one of claims I to 12 or the kit according to any one of claims 2 to 12, wherein the immunogen is (a) adsorbed to the aluminium salt and/or (b) adsorbed to the complex.

14. The immunogenic composition according to any one of claims I to 13 or the kit according to any one of claims 2 to 13,, wherein the immunogen elicits an immune response against a meningococcal bacterium.

15. The immunogenic composition according to any one of claims 1 to 14, or the kit according to any one of claims 2 to 14, wherein the immunogen is from serogroup B meningococcus.

16. The immunogenic composition according to any one of claims I to 13 or the kit according to any one of claims 2 to 13, wherein the immunogen elicits an immune response against an Escherichia coli bacterium.

17. The immunogenic composition according to any one of claim 1 to 16 or the kit according to any one of claims 2 to 16, wherein the adjuvant comprises a buffer and/or 5-15mg/ml sodium chloride.

18. The immunogenic composition or kit according to claim 17, wherein the buffer is a histidine buffer.

19. A method a raising an immune response against a bacterial or fungal pathogen in a subject, said method comprising administering to said subject an immunogenic composition according to any one of claims I to 18 or administering to said subject an immunogenic composition prepared using the kit according to any one of claims 2 to 18.

20. Use of the kit according to any one of claims 2 to 18 in the preparation of a medicament comprising a complex between an immunostimulatory oligonucleotide and a polycationic polymer for treatment or prevention of infection by a bacterial or fungal pathogen. - 33 -

21. The immunogenic composition according to any one of claims 1 to 18 or the kit according to any one of claims 2 to 18 or the method according to claim 19 or the use according to claim 20 substantially as hereinbefore described with reference to the accompanying Figures and/or Examples. DATED this TENTH day of DECEMBER, 2013 Novartis AG By the patent attorneys for the applicant: FB Rice - 34 -